Process of preparing chondrocyte cell suspension and its use

Abstract

A process for the preparation of chondrocyte cell suspension and its use in defect site of knee or ankle or shoulder or wrist or elbow or hip of subject.

1 . A process of preparing chondrocyte cell suspension comprising
 (a) harvesting 40 to 100 mg weight of cartilage tissue from non-weight bearing area of knee of the subject;   (b) mincing the tissue from (a) followed by digesting with enzyme(s) for the isolation of chondrocyte cells;   (c) mixing chondrocyte cells with nutrient medium, serum and optionally growth factors;   (d) optionally seeding to enable cell multiplication until P2 stage to obtain not less than 48 million cells within four weeks;   (e) centrifuging, discarding the supernatant;   (f) mixing with nutrient medium;   (g) analyzing and characterizing the chondrocyte cell suspension;   (h) filling the characterized chondrocyte cell suspension in transparent V shaped 1 ml vials; and optionally transporting to the same subject as in (a).   
     
     
         2 . A process of preparing chondrocyte cell suspension as claimed in  claim 1  wherein the subject is an adult human subject. 
     
     
         3 . A process of preparing chondrocyte cell suspension as claimed in  claim 1  wherein the enzyme is selected from trypsin-EDTA, Collagenase and the like. 
     
     
         4 . A process of preparing chondrocyte cell suspension as claimed in  claim 1  wherein the nutrient medium is selected from IMDM, EMEM, DMEM and the like. 
     
     
         5 . A process of preparing chondrocyte cell suspension as claimed in  claim 1  wherein the growth factors are selected from IGF, TGF, FGF and the like. 
     
     
         6 . A process of preparing chondrocyte cell suspension as claimed in  claim 1  wherein the seeding is done in T-25 flask and/or T-75 and/or T-150 flask and the like. 
     
     
         7 . A process of preparing chondrocyte cell suspension as claimed in  claim 1  wherein the transportation is at 2 to 8 degree centigrade. 
     
     
         8 . A process of preparing chondrocyte cell suspension as claimed in  claim 1  wherein the analysis performed are Appearance, Sterility,  Mycoplasma , Endotoxin, Cell Counting, Cell Viability, Cell Purity Test, Cell Characterization and Karyotyping Analysis. 
     
     
         9 . A process of preparing chondrocyte cell suspension as claimed in  claim 9  wherein cell characterization is by analyzing chondrocytes CD44 +  and CD151 +  marker expressions. 
     
     
         10 . A process of preparing chondrocyte cell suspension as claimed in  claim 1  further comprising optionally mixing with gel while implanting the chondrocyte cell suspension into the defect site of the knee of the subject by using arthroscopy or mini arthrotomy. 
     
     
         11 . A process of preparing chondrocyte cell suspension as claimed in  claim 11  wherein the defect size ranges from 1 to 20 cm 2 . 
     
     
         12 . A process of preparing chondrocyte cell suspension as claimed in  claim 11  wherein the gel is selected from fibrin, thrombin, thermoreversible gel and the like. 
     
     
         13 . A process of preparing chondrocyte cell suspension as claimed in  claim 11  wherein when gel is used implantation is carried out using Y-shaped canula comprising a blunt needle. 
     
     
         14 . A method of implanting chondrocyte cell suspension for autologous chondrocyte transplantation comprising
 (i) drawing 1 ml of nutrient media from vial 1, mixing with fibrin (concentration ranging from 72 to 110 mg) in vial 2, mixing and aspirating the contents into syringe A;   (ii) drawing 1 ml of nutrient media from vial 1, adding to thrombin (concentration of 500 IU/ml) in vial 3 and mixing;   (iii) drawing 0.2 ml from vial 3 and injecting into empty vial 4;   (iv) subsequently drawing 0.4 ml+0.4 ml from vial(s) of chondrocyte cell suspension, injecting into vial 4 and aspirating the contents into syringe B;   (v) placing syringe A and B on the applicator/holder;   (vi) fixing Y-shaped dual syringe applicator comprising a blunt needle to the two syringes; and   (vii) implanting into the defect site of the subject using arthroscopy or mini arthrotomy.   
     
     
         15 . A method of implanting chondrocyte cell suspension as claimed in  claim 14  wherein nutrient medium is selected from IMDM, EMEM, DMEM and the like. 
     
     
         16 . A method of implanting chondrocyte cell suspension as claimed in  claim 14  wherein implantation is carried out using Y-shaped dual syringe applicator comprising a blunt needle. 
     
     
         17 . A method of implanting chondrocyte cell suspension as claimed in  claim 14  wherein chondrocyte cell suspension is prepared by a process as claimed in  claims 1  to  9 . 
     
     
         18 . A method of implanting chondrocyte cell suspension as claimed in  claim 14  wherein the subject is an adult human subject. 
     
     
         19 . A method of implanting chondrocyte cell suspension as claimed in  claim 14  wherein the defect site is in knee or ankle or shoulder or wrist or elbow or hip of subject. 
     
     
         20 . A method of implanting chondrocyte cell suspension as claimed in  claim 14  wherein the chondrocyte cell suspension is prepared by a process comprising
 (a) harvesting 40 to 100 mg weight of cartilage tissue from non-weight bearing area of knee of adult human subject; 
 (b) mincing the tissue from (a) followed by digesting with enzyme(s) for the isolation of chondrocyte cells, wherein the enzyme(s) is selected from trypsin-EDTA, Collagenase and the like; 
 (c) mixing chondrocyte cells with nutrient medium, serum and optionally growth factors, wherein the nutrient medium is selected from IMDM, EMEM, DMEM and the like; 
 (d) optionally seeding to enable cell multiplication until P2 stage to obtain not less than 48 million cells within four weeks, wherein the seeding is done in T-25 flask and/or T-75 and/or T-150 flask and the like; 
 (e) centrifuging, discarding the supernatant; 
 (f) mixing with nutrient medium; 
 (g) analysing and characterizing the chondrocyte cell suspension; wherein analysis performed are Appearance, Sterility,  Mycoplasma , Endotoxin, Cell Counting, Cell Viability, Cell Purity Test, Cell Characterization and Karyotyping Analysis; further wherein cell characterization of chondrocytes is analyzed by CD44 +  and CD151 +  marker expressions; 
 (h) filling the characterized chondrocyte cell suspension in transparent V shaped 1 ml vials; and optionally transporting to the same subject as in (a) at 2 to 8 degree centigrade.